ABSTRACT The goal of this project is to bring a new optical imaging technology to the biomedical community, and establish it as a simple, versatile and robust alternative to confocal microscopy. Our technique is called HiLo imaging, and was invented by our lab in the summer of 2008. HiLo imaging allows confocal-like out-of-focus fluorescence background rejection without the use of a confocal scanning mechanism. In brief, it requires the acquisition of two images, one with structured illumination and another with standard uniform illumination. HiLo imaging works with both fluorescent and non-fluorescent samples. Since it is based on conventional widefield imaging, it can readily be adapted to standard microscopes. Moreover, since it requires only two images, it can be operated at very high acquisition rates that easily surpass video rate (to be demonstrated). This project is focused mostly on instrumentation development. Having already established the basic principles of HiLo microscopy, we plan to experimentally quantify key performance parameters, including axial resolution, signal to noise ratio and depth penetration. To demonstrate the versatility of HiLo imaging and its broad range of applicability, we plan to confirm the effectiveness of HiLo imaging in situations where confocal microscopy is impractical or impossible. Specifically, we plan to build both a HiLo endomicroscope and a HiLo macroscope. The former will be used to perform intravital confocal-like imaging in rat colons, in collaboration with the Dr. Satish Singh group at the Boston University School of Medicine Gastroenterology Department, with the goal of demonstrating the effectiveness of HiLo endomicroscopy for optical biopsy and cancer diagnosis. Our second device, a HiLo macroscope, will exploit the versatility of HiLo imaging by providing what confocal microscopy cannot, namely video-rate, out-of-focus background rejection over a very wide field of view (about 1cm) and with a long working distance (several cms). The applications of this macroscope will be in molecular imaging of small animals, and we propose to demonstrate its effectiveness at characterizing abnormal vasculature in mouse tumor models, in collaboration with the Dr. Rakesh Jain group at the Massachusetts General Hospital Department of Radiation Oncology. HiLo imaging is a new technology. Our goal is to firmly establish the advantages of this technology so that it will become routine instrumentation in biomedical imaging.